1. Field of the Invention
The present invention relates to methods of detecting G-protein-coupled receptor (GPCR) activity, and provides methods of assaying GPCR activity, methods for screening for GPCR ligands, agonists and/or antagonists, methods for screening natural and surrogate ligands for orphan GPCRs, and methods for screening compounds that interact with components of the GPCR regulatory process.
2. Background of the Technology
The actions of many extracellular signals are mediated by the interaction of G-protein-coupled receptors (GPCRs) and guanine nucleotide-binding regulatory proteins (G-proteins). G-protein-mediated signaling systems have been identified in many divergent organisms, such as mammals and yeast. The GPCRs represent a large super family of proteins which have divergent amino acid sequences, but share common structural features, in particular, the presence of seven transmembrane helical domains. GPCRs respond to, among other extracellular signals, neurotransmitters, hormones, odorants and light. Individual GPCR types activate a particular signal transduction pathway; at least ten different signal transduction pathways are known to be activated via GPCRs. For example, the beta 2-adrenergic receptor (xcex22AR) is a prototype mammalian GPCR. In response to agonist binding, xcex22AR receptors activate a G-protein (Gs) which in turn stimulates adenylate cyclase activity and results in increased cyclic adenosine monophosphate (cAMP) production in the cell.
The signaling pathway and final cellular response that result from GPCR stimulation depends on the specific class of G-protein with which the particular receptor is coupled (Hamm, xe2x80x9cThe Many Faces of G-Protein Signaling.xe2x80x9d J. Biol. Chem., 273:669-672 (1998)). For instance, coupling to the Gs class of G-proteins stimulates cAMP production and activation of the Protein Kinase A and C pathways, whereas coupling to the Gi class of G-proteins down regulates cAMP. Other second messenger systems such as calcium, phospholipase C, and phosphatidylinositol 3 may also be utilized. As a consequence, GPCR signaling events have predominantly been measured via quantification of these second messenger products.
The decrease of a response to a persistent stimulus is a widespread biological phenomenon. Signaling by diverse GPCRs is believed to be terminated by a uniform two-step mechanism. Activated receptor is first phosphorylated by a GPCR kinase (GRK). An arrestin protein binds to the activated and phosphorylated receptor, thus blocking G-protein interaction. This process is commonly referred to as desensitization, a general mechanism that has been demonstrated in a variety of functionally diverse GPCRs. Arrestin also plays a part in regulating GPCR internalization and resensitization, processes that are heterogenous among different GPCRs (Oakley, et al., J. Biol. Chem., 274:32248-32257 (1999)). The interaction between an arrestin and GPCR in processes of internalization and resensitization is dictated by the specific sequence motif in the carboxyl terminus of a given GPCR. Only a subset of GPCRs, which possess clusters of three serine or threonine residues at the carboxyl termini, were found to co-traffick with the arrestins into the endocytic vesicles after ligand stimulation. The number of receptor kinases and arrestins involved in desensitization of GPCRs is rather limited.
A common feature of GPCR physiology is desensitization and recycling of the receptor through the processes of receptor phosphorylation, endocytosis and dephosphorylation (Ferguson, et al., xe2x80x9cG-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins.xe2x80x9d Can. J. Physiol. Pharmacol., 74:1095-1110 (1996)). Ligand-occupied GPCRs can be phosphorylated by two families of serine/threonine kinases, the G-protein-coupled receptor kinases (GRKs) and the second messenger-dependent protein kinases such as protein kinase A and protein kinase C. Phosphorylation by either class of kinases serves to down-regulate the receptor by uncoupling it from its corresponding G-protein. GRK-phosphorylation also serves to down-regulate the receptor by recruitment of a class of proteins known as the arrestins that bind the cytoplasmic domain of the receptor and promote clustering of the receptor into endocytic vescicles. Once the receptor is endocytosed, it will either be degraded in lysosomes or dephosphorylated and recycled back to the plasma membrane as a fully-functional receptor.
Binding of an arrestin protein to an activated receptor has been documented as a common phenomenon of a variety of GPCRs ranging from rhodopsin to xcex22AR to the neurotensin receptor (Barak, et al., xe2x80x9cA xcex2-arrestin/Green Fluorescent Fusion Protein Biosensor for Detecting G-Protein-Coupled Receptor Activation,xe2x80x9d J. Biol. Chem., 272:27497-500 (1997)). Consequently, monitoring arrestin interaction with a specific GPCR can be utilized as a generic tool for measuring GPCR activation. Similarly, a single G-protein and GRK also partner with a variety of receptors (Hamm, et al. (1998) and Pitcher et al., xe2x80x9cG-Protein-Coupled Receptor Kinases,xe2x80x9d Annu. Rev. Biochem., 67:653-92 (1998)), such that these protein/protein interactions may also be monitored to determine receptor activity.
Many therapeutic drugs in use today target GPCRs, as they regulate vital physiological responses, including vasodilation, heart rate, bronchodilation, endocrine secretion and gut peristalsis. See, e.g., Lefkowitz et al., Annu. Rev. Biochem., 52:159 (1983). Some of these drugs mimic the ligand for this receptor. Other drugs act to antagonize the receptor in cases when disease arises from spontaneous activity of the receptor.
Efforts such as the Human Genome Project are identifying new GPCRs (xe2x80x9corphanxe2x80x9d receptors) whose physiological roles and ligands are unknown. It is estimated that several thousand GPCRs exist in the human genome.
Various approaches have been used to monitor intracellular activity in response to a stimulant, e.g., enzyme-linked immunosorbent assay (ELISA); Fluorescense Imaging Plate Reader assay (FLIPR(trademark), Molecular Devices Corp., Sunnyvale, Calif.); EVOscreen(trademark), EVOTEC(trademark), Evotec Biosystems Gmbh, Hamburg, Germany; and techniques developed by CELLOMICS(trademark), Cellomics, Inc., Pittsburgh, Pa.
Germino et al., xe2x80x9cScreening for in vivo protein-protein interactions.xe2x80x9d Proc. Natl. Acad. Sci., 90(3):933-937 (1993), discloses an in vivo approach for the isolation of proteins interacting with a protein of interest.
Phizicky et al., xe2x80x9cProtein-protein interactions: methods for detection and analysis.xe2x80x9d Microbiol. Rev., 59(1): 94-123 (1995), discloses a review of biochemical, molecular biological and genetic methods used to study protein-protein interactions.
Offermanns et al., xe2x80x9cGxcex115 and Gxcex116 Couple a Wide Variety of Receptors to Phospholipase C.xe2x80x9d J. Biol. Chem., 270(25):15175-15180 (1995), discloses that Gxcex115 and Gxcex116 can be activated by a wide variety of G-protein-coupled receptors. The selective coupling of an activated receptor to a distinct pattern of G-proteins is regarded as an important requirement to achieve accurate signal transduction. Id.
Barak et al., xe2x80x9cA xcex2-arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation.xe2x80x9d J. Biol. Chem., 272(44):27497-27500 (1997) and U.S. Pat. Nos. 5,891,646 and 6,110,693 disclose the use of a xcex2-arrestin/green fluorescent fusion protein (GFP) for imaging protein translocation upon stimulation of GPCR with optical devices.
Each of the references described above has drawbacks. For example,
The prior art methodologies require over-expression of the proteins, which could cause artifact and tip the balance of cellular regulatory machineries.
The prior art visualization or imaging assays are low throughput and lack thorough quantification. Therefore, they are not suitable for high throughput pharmacological and kinetic assays.
In addition, many of the prior art assays require isolation of the GPCR rather than observation of the GPCR in a cell. There thus exists a need for improved methods for monitoring GPCR function.
The present invention provides modifications to the disclosure in U.S. application Ser. No. 09/654,499. In particular, the present invention is directed to modifications of the below aspects of the invention to further enhance assay sensitivity. The modifications include the use of genetically modified arrestins that exhibit enhanced binding to activated GPCR regardless of whether the GPCR is phosphorylated or non-phosphorylated; the use of a serine/threonine cluster strategy to facilitate screening assays for orphan receptors that do not possess this structural motif on their own; and the use of a combination of the above modifications to achieve even more enhanced detection.
A first aspect of the present invention is a method that monitors GPCR function proximally at the site of receptor activation, thus providing more information for drug discovery purposes due to fewer competing mechanisms. Activation of the GPCR is measured by a read-out for interaction of the receptor with a regulatory component such as arrestin, G-protein, GRK or other kinases, the binding of which to the receptor is dependent upon agonist occupation of the receptor. The present invention involves the detection of protein/protein interaction by complementation of mutant reporter enzymes.
Binding of arrestin to activated GPCR is a common process in the first step of desensitization that has been demonstrated for most, if not all, GPCRs studied so far. Measurement of GPCR interaction with arrestin via mutant enzyme complementation (i.e., ICAST) provides a more generic assay technology applicable for a wide variety of GPCRs and orphan receptors.
A further aspect of the present invention is a method of assessing GPCR pathway activity under test conditions by providing a test cell that expresses a GPCR, e.g., muscarinic, adrenergic, dopamine, angiotensin or endothelin, as a fusion protein to a mutant reporter enzyme and interacting a protein in the GPCR pathway, e.g., G-protein, arrestin or GRK, as a fusion protein with a complementing mutant reporter enzyme. When test cells are exposed to a known agonist to the target GPCR under test conditions, activation of the GPCR will be monitored by complementation of the reporter enzyme. Increased reporter enzyme activity reflects interaction of the GPCR with its interacting protein partner.
A further aspect of the present invention is a method of assessing GPCR pathway activity in the presence of a test arrestin, e.g., xcex2-arrestin.
A further aspect of the present invention is a method of assessing GPCR pathway activity in the presence of a test G-protein.
A further aspect of the present invention is a method of assessing GPCR pathway activity upon exposure of the test cell to a test ligand.
A further aspect of the present invention is a method of assessing GPCR activity upon co-expression in the test cell of a second receptor. The second receptor could be the same GPCR or orphan receptor (i.e., homo-dimerization), a different GPCR or orphan receptor (i.e., hetero-dimerization) or could be a receptor of another type.
A further aspect of the present invention is a method for screening for a ligand or agonist to an orphan GPCR. The ligand or agonist could be contained in natural or synthetic libraries or mixtures or could be a physical stimulus. A test cell is provided that expresses the orphan GPCR as a fusion protein with a mutant reporter enzyme, e.g., a xcex2-galactosidase mutant, and, for example, an arrestin or mutant form of arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another xcex2-galactosidase mutant. The interaction of the arrestin with the orphan GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of a ligand or agonist.
A further aspect of the present invention is a method for screening a protein of interest, for example, an arrestin protein (or mutant form of the arrestin protein) for the ability to bind to a phosphorylated, or activated, GPCR. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a xcex2-galactosidase mutant, and contains arrestin (or a mutant form of arrestin) as a fusion protein with a complementing mutant reporter enzyme, e.g., another xcex2-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a known GPCR agonist and then reporter enzyme activity is detected. Increased reporter enzyme activity indicates that the xcex2-arrestin molecule can bind to phosphorylated, or activated, GPCR in the test cell.
A further aspect of the present invention is a method to screen for an agonist to a specific GPCR. The agonist could be contained in natural or synthetic libraries or could be a physical stimulus. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a xcex2-galactosidase mutant, and, for example, an arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another xcex2-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of an agonist. The test cell may express a known GPCR or a variety of known GPCRs, or may express an unknown GPCR or a variety of unknown GPCRs. The GPCR may be, for example, an odorant GPCR or a xcex2AR GPCR.
A further aspect of the present invention is a method for screening a test compound for GPCR antagonist activity. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a xcex2-galactosidase mutant, and, for example, an arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another xcex2-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of an agonist. The cell is exposed to a test compound and to a GPCR agonist, and reporter enzyme activity is detected. When exposure to the agonist occurs at the same time as or subsequent to exposure to the test compound, a decrease in reporter enzyme activity after exposure to the test compound indicates that the test compound has antagonist activity to the GPCR.
A further aspect of the present invention is a method of screening a sample solution for the presence of an agonist, antagonist or ligand to a GPCR. A test cell is provided that expresses GPCR as a fusion protein with a mutant reporter enzyme, e.g., a xcex2-galactosidase mutant, and contains, for example, a xcex2-arrestin as a fusion protein with a complementing reporter, e.g., another xcex2-galactosidase mutant. The test cell is exposed to a sample solution, and reporter enzyme activity is assessed. Changed reporter enzyme activity after exposure to the sample solution indicates the sample solution contains an agonist, antagonist or ligand for a GPCR expressed in the cell.
A further aspect of the present invention is a method of screening a cell for the presence of a GPCR. According to this aspect, an arrestin fusion protein with a mutant reporter enzyme and a GPCR downstream signaling fusion protein with a mutant reporter enzyme are employed to detect GPCR action. A modification of this aspect of the invention can be employed to provide a method of screening a plurality of cells for those cells which contain a GPCR. According to this aspect, a plurality of cells containing a conjugate comprising a xcex2-arrestin protein as a fusion protein with a reporter enzyme are provided; the plurality of cells are exposed to a GPCR agonist; and activity of reporter enzyme activity is detected. An increase in reporter enzymatic activity after exposure to the GPCR agonist indicates xcex2-arrestin protein binding to a GPCR, thereby indicating that the cell contains a GPCR responsive to the GPCR agonist.
A further aspect of the invention is a method for mapping GPCR-mediated signaling pathways. For instance, the system could be utilized to monitor interaction of c-src with xcex2-arrestin-1 upon GPCR activation. Additionally, the system could be used to monitor protein/protein interactions involved in cross-talk between GPCR signaling pathways and other pathways such as that of the receptor tyrosine kinases or Ras/Raf. According to this aspect, a test cell is provided that expresses a GPCR or other related protein with a mutant reporter enzyme, e.g., a xcex2-galactosidase mutant, and contains a protein from another pathway as a fusion protein with a complementing mutant reporter enzyme, e.g., another xcex2-galactosidase mutant. Increased reporter enzymatic activity indicates protein/protein interaction.
A further aspect of the invention is a method for monitoring homo- or hetero-dimerization of GPCRs upon agonist or antagonist stimulation. Increasing evidence indicates that GPCR dimerization is important for biological activity (AbdAlla, et al., xe2x80x9cAT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration.xe2x80x9d Nature, 407:94-98 (2000); Bockaert, et al., xe2x80x9cMolecular tinkering of G protein-coupled receptors: an evolutionary success.xe2x80x9d EMBO J. 18:1723-29 (1999)). Jordan, et al., xe2x80x9cG-protein-coupled receptor heterodimerization modulates receptor function.xe2x80x9d Nature, 399:697-700 (1999), demonstrated that two non-functional opioid receptors, xcexa and xcex4, heterodimerize to form a functional receptor. Gordon et al., xe2x80x9cDopamine D2 receptor dimers and receptor blocking peptides.xe2x80x9d Bioch. Biophys. Res. Commun. 227:200-204 (1996), showed different pharmacological properties associated with the monomeric and dimeric forms of Dopamine receptor D2. The D2 receptors exist either as monomers that are selective targets for spiperone or as dimer forms that are targets for nemonapride. Herbert, et al., xe2x80x9cA peptide derived from a xcex22-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation.xe2x80x9d J.B.C. 271:16384-92 (1996), demonstrated that the agonist stimulation was found to stabilize the dimeric state of the receptor, whereas inverse agonists favored the monomeric form. Indeed, the same study showed that a peptide corresponding to the sixth transmembrane domain of the xcex22-adrenergic receptor inhibited both receptor dimerization and activation. Further, Angers et al., Detection of beta-2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer, Proc. Natl. Acad. Sci. USA, 97(7):3684-3689, discloses the use of xcex22-adrenergic receptor fusion proteins (i.e., xcex22-adrenergic receptor fused to luciferase and xcex22-adrenergic receptor fused to an enhanced red-shifted green fluorescent protein) to study xcex22-adrenergic receptor dimerization.
GPCR dimerization in the context of cellular physiology and pharmacology can be monitored in accordance with the invention. For example, xcex2-galactosidase complementation can be measured in test cells that co-express GPCR fusion proteins of xcex2-galactosidase mutant enzymes, e.g., GPCR1xcex94xcex1 and GPCR2xcex94xcfx89 (FIG. 27). According to this aspect, the interconversion between monomeric to dimeric forms of the GPCRs or orphan receptors can be measured by mutant reporter enzyme complementation. FIG. 27 illustrates a test cell co-expressing GPCR or an orphan receptor as a fusion protein with xcex94xcex1 form of xcex2-galactosidase mutant (e.g., GPCR1xcex94xcex1), and the same GPCR or orphan receptor as a fusion protein with xcex94xcfx89 form of xcex2-galactosidase mutant (e.g. GPCR1xcex94xcfx89). Formation of the GPCR homodimer is reflected by formation of an active enzyme, which can be measured by enzyme activity assays, such as the Gal-Screen(trademark) assay. Similarly, hetero-dimerization between two distinct GPCRs, or two distinct orphan receptors, or between one known GPCR and one orphan receptor can be analyzed in test cells co-expressing two fusion proteins, e.g., GPCR1xcex94xcex1 and GPCR2xcex94xcfx89. The increased xcex2-galactosidase activity indicates that the two receptors can form a heterodimer.
A further aspect of the invention is a method of monitoring the interconversion between the monomeric and dimeric form of GPCRs under the influence of agonist or antagonist treatment. The test receptor(s) can be between the same GPCR or orphan receptor (homodimer), or between two distinct GPCRs or orphan receptors (heterodimer). The increased xcex2-galactosidase activity after treatment with a compound means that the compound binds to and/or stabilizes the dimeric form of the receptor. The decreased xcex2-galactosidase activity after treatment with a compound means that the compound binds to and/or stabilizes the monomeric form of the receptor.
A further aspect of the invention is a method of screening a cell for the presence of a GPCR responsive to a GPCR agonist. A cell is provided that contains protein partners that interact downstream in the GPCR""s pathway. The protein partners are expressed as fusion proteins to the mutant, complementing enzyme and are used to monitor activation of the GPCR. The cell is exposed to a GPCR agonist and then enzymatic activity of the reporter enzyme is detected. Increased reporter enzyme activity indicates that the cell contains a GPCR responsive to the agonist.
The present invention involves the use of a combination of proprietary technologies (including ICAST(trademark), Intercistronic Complementation Analysis Screening Technology, Gal-Screen(trademark), etc.) to monitor protein/protein interactions in GPCR signaling. As disclosed in U.S. application Ser. No. 09/654,499, the method of the invention in part involves using ICAST(trademark), which in turn involves the use of two inactive xcex2-galactosidase mutants, each of which is fused with one of two interacting target protein pairs, such as a GPCR and an arrestin. The formation of an active xcex2-galactosidase complex is driven by interaction of the target proteins. In this system, xcex2-galactosidase activity can be detected using, e.g., the Gal-Screen(trademark) assay system, wherein direct cell lysis is combined with rapid ultrasensitive chemiluminescent detection of xcex2-galactosidase reporter enzyme. This system uses, e.g., a Galacton-Star(copyright) chemiluminescent substrate for measurement in a luminometer as a read out of GPCR activity.
FIG. 23 is a schematic depicting the use of the complementation technology in the method of the present invention. FIG. 23 shows two inactive xcex2-galactosidase mutants that become active when they are forced together by specific interactions between the fusion partners of an arrestin molecule and an activated GPCR or orphan receptor. This assay technology will be especially useful in high throughput screening assays for ligand fishing for orphan receptors, a process called de-orphaning. As illustrated in FIG. 28, a xcex2-galactosidase fusion protein of an orphan receptor (e.g., GPCRorphanxcex94xcex1) is co-expressed in the test cell with a fusion protein of xcex2-arrestin (e.g., xcex2-Arrxcex94xcfx89). When the test cell is subjected to compounds, which could be natural or synthetic, the increased xcex2-galactosidase activity means the compound is either a natural or surrogate ligand for this GPCR. The same assay system can be used to find drug leads for the new GPCRs. The increased xcex2-galactosidase activity in the test cell after treatment indicates the agonist activity of the compound. The decreased xcex2-galactosidase activity in the test cell indicates antagonist activity or inverse agonist activity of the compound. In addition, the method of the invention could be used to monitor GPCR-mediated signaling pathways via other downstream signaling components such as G-proteins, GRKs or the proto-oncogene c-Src.
The invention is achieved in part by using ICAST(trademark) protein/protein interaction screening to map signaling pathways. This technology is applicable to a variety of known and unknown GPCRs with diverse functions. They include, but are not limited to, the following sub-families of GPCRs:
(a) receptors that bind to amine-like ligands-Acetylcholine muscarinic receptor (M1 to M5), alpha and beta Adrenoceptors, Dopamine receptors (D1, D2, D3 and D4), Histamine receptors (H1 and H2), Octopamine receptor and Serotonin receptors (5HT1, 5HT2, 5HT4, 5HT5, 5HT6, 5HT7);
(b) receptors that bind to a peptide ligand-Angiotensin receptor, Bombesin receptor, Bradykinin receptor, Cxe2x80x94C chemokine receptors (CCR1 to CCR8, and CCR10), C-X-C type Chemokine receptors (CXC-R5), Cholecystokinin type A receptor, CCK type receptors, Endothelin receptor, Neurotesin receptor, FMLP-related receptors, Somatostatin receptors (type 1 to type 5) and Opioid receptors (type D, K, M, X);
(c) receptors that bind to hormone proteins-Follic stimulating hormone receptor, Thyrotrophin receptor and Lutropin-choriogonadotropic hormone receptor;
(d) receptors that bind to neurotransmitters-substance P receptor, Substance K receptor and neuropeptide Y receptor;
(e) Olfactory receptors-Olfactory type 1 to type 11, Gustatory and odorant receptors;
(f) Prostanoid receptors-Prostaglandin E2 (EP1 to EP4 subtypes), Prostacyclin and Thromboxane;
(g) receptors that bind to metabotropic substances-Metabotropic glutamate group I to group III receptors;
(h) receptors that respond to physical stimuli, such as light, or to chemical stimuli, such as taste and smell; and
(i) orphan GPCRs-the natural ligand to the receptor is undefined.
Use of the ICAST(trademark) technology in combination with the invention provides many benefits to the GPCR screening process, including the ability to monitor protein interactions in any sub-cellular compartment-membrane, cytosol and nucleus; the ability to achieve a more physiologically relevant model without requiring protein overexpression; and the ability to achieve a functional assay for receptor binding allowing high information content.